Biosensor

ABSTRACT

A sensor for determining the presence of an analyte in a test sample, said sensor comprising a nanoparticulate membrane comprising nanoparticles of at least one inorganic oxide of an element selected from Group IA, IIA, IIIA, IVA, IB, IIB, IIIB, IVAB, VB, VIB, VIII3 or VIIII3 of the Periodic Table, and wherein an oxidoreductase and an electrochemical activator are diffusibly dispersed in said nanoparticulate membrane.

The present invention relates to sensors, in particular sensors fordetermining the presence of analytes in a test sample. The inventionalso relates to nanoparticulate membranes. The invention further relatesto water-soluble redox polymers and processes for preparing thepolymers.

BACKGROUND OF THE INVENTION

In recent years, polymeric materials have gained widespread theoreticalinterest and practical use in many fields [G. Harsanyi. MaterialsChemistry and Physics Vol. 43, Issue 3, 1996, 199]. Conducting polymersin particular have found increasing use in the field of biosensing,where conducting polymers provide a unique function as an interfacebetween smart sensors and intelligent molecular receptors. Of all theknown conducting polymers, such as ionically conducting polymers, chargetransfer polymers and conjugated conducting polymers, redox polymers areby far most widely used in biosensing applications.

Glucose sensing, an area in biosensing which has been undergoingsignificant research in recent years, relies on electron mediation ofenzymatic oxidation of glucose to gluconic acid by glucose oxidase isrequired. The electron mediating function of redox polymers has beenwidely studied and applied to many amperometric glucose biosensors.

In its natural enzymatic reaction, co-enzyme flavin adenine dinucleotide(FAD) is an electron carrier present in glucose oxidase is reduced toFADH₂ (reduced form of FAD) and oxidized back to FAD by molecularoxygen. O₂ is then reduced to H₂O₂. This cyclic oxidation and reductionenables FAD to act as an electron acceptor. Since neither glucose norgluconic acid is electro-active within the working potential window from−0.5 to 1.0 V, either the increase in H₂O₂ concentration or the decreasein O₂ concentration is being measured to quantify the glucoseconcentration.

However, the accuracy of measurements based on the measurement of H₂O₂and O₂ is compromised because firstly, the partial pressure ofatmospheric O₂ affects amperometric response, and secondly, thequantitative measurement of O₂ at high glucose concentration isdifficult because O₂ is used up as the sensing proceeds. The detectionof H₂O₂ by its oxidation at a platinum electrode requires a workingpotential of 0.5 to 0.6 V (vs. Ag/AgCl), and thus is subjected tointerferences of electro-active species in blood, such as ascorbic acidand uric acid which are electrochemically active at this potential.

To circumvent the above-mentioned problems associated with glucosemonitoring involving O₂ or H₂O₂, redox-active mediators have beenproposed as artificial electron acceptors in place of oxygen moleculesfor FADH₂.

A successful mediator should, in principle, meet three requirements: (1)fast electron-exchange rate with enzyme and electrode, (2) stableattachment to the electrode and (3) processable in aqueous medium.

For this reason, two groups of mediators were extensively investigated,namely, transition metal complexes and ferrocenyl materials. In recentyears, many groups have focused their attention on the synthesis andbiosensing applications of ferrocenyl materials, both monomeric andpolymeric. For example, polyferrocenyl compounds have been used as redoxindicators in molecular recognition [J. E. Kinston, et al, J. Chem.Soc., Dalton. Trans (1999) 251.], as mediators in biosensors [S. Koide,et al, J. Electroanal. Chem., 468(1999) 193.] and as coating to modifiedelectrode surface [S. Niate, et al, Chem. Commun., (2000) 417.].However, most of the known ferrocenyl materials are only soluble innon-polar media, only few ferrocenyl and polyferrocenyl materials arewater-soluble [O. Hatozaki, et al, J. Phys. Chem., 199 (1996) 8448.].Water-soluble ferrocenyl materials are of particular interest as redoxmediators in biosensing. By co-polymerizing alkene substitutedferrocenes, such as vinylferrocene, with an appropriate water-solublepolymer, it is possible to prepare ferrocenyl materials that are readilysoluble in water. But it has been shown that the free radical initiatedpolymerization of vinylferrocene is unusual [A. J. Tinker, et al, J.Polym Sci., Polym. Chem. Ed., 13 (1975) 2133; M. H. George, et al, J.Polym Sci., Polym. Chem. Ed., 14 (1975) 475.]. Co-polymerization ofvinylferrocene is known to be difficult because the ferrocenium is aradical scavenger in the polymerization system, resulting in that thereaction does not obey normal radical polymerization kinetics.Termination of the polymerization reaction occurs by an intramolecularelectron transfer from a ferrocene nucleus to the growing chain radical.This leads to the deactivation of the polymer chain and a polymer whichcontains a high spin Fe(III) species.

Polyacrylamide has been widely used as support matrix in enzymeimmobilization and biosensing because of its good chemical andmechanical stability and its inertness to microbial degradation [I,Willner, et al, J. Am. Chem. Soc., 112 (1990) 6438.]. However, attemptsof co-polymerization of vinylferrocene and acrylamide and itsderivatives were not successful [H. Bu, et al, Anal. Chem., 67 (1995)4071 and references therein.]. Instead, to by-pass the inefficientco-polymerization of vinylferrocene, chemical grafting procedures wereproposed in preparing ferrocenyl materials [S. Koide, et al, J.Electroanal. Chem., 468 (1999) 193; J. Hodak, et al, Langmuir, 13 (1997)2708; A. Salmon, et al, J. Organomet. Chem., 637-639 (2001) 595.]. Intwo recent reports [N. Kuramoto, et al, Polymer 39 (1998) 669; H. Ahmad,et al, Colloids and Surfaces, 186 (2001) 221], vinylferroceneco-polymers were synthesized, but minute loading of ferrocene and lackof cross-linkable groups in these polymers restrict their use inbiosensors.

Commercially available biosensors include those manufactured byTherasense Inc. (cf., for example U.S. Pat. No. 6,338,790), InvernessMedical Technology (cf., for example U.S. Pat. No. 6,241,862) andMatsushita Electric (cf., for example, U.S. Pat. No. 6,547,954).

Therefore, there remains the need for vinylferrocene-based polymericmediators having superior performance characteristics. Consequently, itis a goal of the present invention to develop new methods of synthesisfor new vinylferrocene-based polymeric mediators. It is also a goal ofthis invention to provide biosensors with enhanced performance, andwhich would impose minimal inconvenience to the end user of thebiosensor as much as possible.

These goals are solved by the various aspects of the present invention,namely the sensors, membranes, polymers, and processes as defined in therespective independent claims.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a sensor for determining thepresence of an analyte in a test sample, said sensor comprising ananoparticulate membrane comprising nanoparticles of at least oneinorganic oxide of an element selected from Group IA, IIA, IIIA, IVA,IB, IIB, IIIB, IVAB, VB, VIB, VIIB or VIIIB of the Periodic Table, andwherein an oxidoreductase enzyme and electrochemical activator arediffusibly dispersed in said nanoparticulate membrane.

In yet another aspect, the invention provides an electricallynon-conductive, nanoparticulate membrane comprising nanoparticles of atleast one inorganic oxide of an element selected from Group IA, IIA,IIIA, IVA, IB, IIB, IIIB, IVAB, VB, VIB, VIIB or VIIIB of the PeriodicTable, and wherein an oxidoreductase enzyme and electrochemicalactivator are diffusibly dispersed in said nanoparticulate membrane.

In a further aspect the invention provides a process for producing anelectrically non-conductive, nanoparticulate membrane comprising mixingan electrochemical redox mediator with an oxidoreductase enzyme andnanoparticles of an oxide of an element selected from Group IA, IIA,IIIA, IVA, IB, IIB, IIIB, IVAB, VB, VIB, VIIB or VIIIB to form ananocomposite ink; and applying said nanocomposite ink onto a substrate.

In yet another aspect, the invention provides a water soluble redoxpolymer comprising:

-   -   a first monomer unit comprising a polymerisable ferrocene        derivative; and    -   a second monomer unit comprising an acrylic acid derivative        having a (terminal) primary acid or base acid or base functional        group capable of acquiring a net charge.

In one embodiment, the acrylic acid derivative in this new water solubleredox polymer is represented by the general formula (I)

wherein R is selected from the group consisting of C_(n)H_(2n)—NH₂,C_(n)H_(2n)—COOH, NH—C_(n)H_(2n)—PO₃H and NH—C_(n)H_(2n)—SO₃H, whereinthe alkyl chain can be optionally substituted, and wherein n is aninteger from 0 to 12.

In yet another aspect, the invention provides a process for preparing awater soluble, redox polymer, said process comprising:

-   -   polymerising a first monomer unit comprising a polymerisable        ferrocene derivative with a second monomer unit comprising an        acrylic acid derivative having an acid or base functional group        capable of acquiring a net charge, wherein said polymerization        is carried out in an aqueous alcoholic medium.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the detaileddescription when considered in conjunction with the non-limitingexamples and the drawings, in which:

FIG. 1 is an exploded isometric drawing of a biosensor according to anembodiment of the invention.

FIG. 2A is a drawing showing an end view of the biosensor in FIG. 1, asseen in the direction of an arrow A in FIG. 1.

FIG. 2B is a drawing showing another end view of the biosensor in FIG.1, as seen in the direction of an arrow B in FIG. 1.

FIG. 3 is an exploded isometric drawing of a biosensor according toanother embodiment of the invention.

FIG. 4 shows a schematic diagram of the coupling redox reaction whichtakes places in a redox polymer mediated biosensor.

FIG. 5 illustrates a structure of the basic unit of a water-soluble andcross-linkable polymer of the present invention. The figure shows arepeating unit found in a copolymer of vinylferrocene and an acrylicacid derivative.

FIG. 6 depicts the general reaction equation in the co-polymerizationreaction of vinyl ferrocene and an acrylic acid derivative.

FIG. 7 shows a Fourier Transform Infra Red (FT-IR) spectrum of the redoxpolymers PAA-VFc and PAAS-VFc produced according to a process of theinvention.

FIG. 8 shows an Ultra Violet (UV)-visible spectra of Fc, PAA, PAAS andthe co-polymers obtained from copolymerization with VFc.

FIG. 9 shows cyclic voltammograms of redox polymers in various systems.Phosphate-buffered saline was used, and the potential scan rate appliedin obtaining the voltammograms was 100 mV/s.

FIG. 10 shows another cyclic voltammogram of a redox polymer PAA-VFcthat is cross-linked with glucose oxidase-bovine serum albumin (GOx-BSA)film on gold electrode. Phosphate-buffered saline was used, and thepotential scan rate applied in obtaining the voltammograms was 50 mV/s.

FIG. 11 shows an exploded view of a disposable glucose biosensor used inthis work.

FIG. 12 shows a cyclic voltammograms of the nanoparticulate printing ink(a) without and (b) with the addition of 100 mg/dl glucose. Potentialscan rate which was applied was 100 mV/s.

FIG. 13 shows amperometric responses of the biosensor in PBS containing(a) 200 and (b) 0.0 mg/dl glucose. The poised potential applied was 0.30V.

FIG. 14 shows dependence of amperometric peak current of 300 mg/dlglucose on (a) PVFcAA concentration, (b) GOX concentration, (c) poisepotential and (d) the nanoparticulate membrane thickness.

FIG. 15 shows amperometric responses of 200 mg/dl glucose in PBS in the(a) absence and (b) presence of dissolved oxygen. The poised potentialapplied was 0.30 V.

FIG. 16 shows (a) amperometric responses to sequential increases of 100mg/dl glucose in PBS and (b) calibration curves at different samplingtimes. The poised potential applied was 0.30 V.

DETAILED DESCRIPTION

In one aspect, the present invention is based on the finding thatwater-soluble and cross-linkable redox polymers can be readily preparedin a mixture of ethanol and water with a persulfate salt as radicalinitiator. This method of preparation allowed problems faced earliersuch as the unfavorable energetics of copolymerization reactionsinvolving ferrocene molecules to be overcome. Tests showed that themolecular weight of vinylferrocene-co-acrylamide copolymers obtainedfrom this method was in the range of 2000 to 4000 Daltons, correspondingto about 400 monomeric units with 3-14% ferrocene loading. Such a levelof ferrocene loading was previously achievable using free radicalpolymerization techniques (see for example N. Kuramoto et al). Byovercoming this limitation, the present invention has given rise to newpolymers with useful properties for biosensing applications and othermethods of electrochemical detection of analytes.

The ferrocene centers in redox polymers of the invention are able toprovide localized electroactivity and thus the ability to engage inredox reactions without bringing about a reorganization of intramolecular bonds in the polymer. Furthermore, redox polymers of theinvention comprise side chains with functional groups that facilitatecross-linking with other molecules with suitable functional groups. Thisallows the polymer to be attached to a wide variety of molecules aswell. Combining these two characteristics, these polymers are welladapted for use in applications requiring electron mediation, such asenzyme electrodes used in biosensors and biofuel cells, as well asenzymatic synthesis carried out in electroenzyme reactors.

In another aspect, glucose sensors according to the present inventionincorporate sensing elements that are capable of accurately measuringthe concentration of glucose found in very small quantities of fluid, sothat test samples of less than 1 μL, or preferably about 0.2 μL to 0.3μL, is needed. Test samples typically include animal biological samplessuch as biological fluids (e.g. blood samples, sweat samples, urinesamples); faecal samples, and flesh samples containing adipose tissue orsubcutaneous fat. Other samples that can be analysed using the presentsensor include reagents utilised in scientific experiments, or food withglucose content and fermentation broths found in the wine or beerproduction industries. Test samples can also include microbiologicalculture mediums (e.g. a growth medium for high density fermentation ofE. coli, yeast or other host organisms, typically used for recombinantproduction of polypeptides).

Due to the small volume of test sample that is required for performing adiagnostic test, minimal inconvenience is imposed on the end user. Forexample, for diabetics that require continuous blood glucose assessment,withdrawal of a blood sample at a sub-microlitre level would imposeminimal pain and disturbance to the patient.

In general, a sensor of the present invention makes use of annanoparticulate membrane which is described herein in detail. Inaddition to the fact that it is suitable for testing of blood sample ata sub-microlitre level, such a membrane has the advantage that is can beproduced at low cost, and is stable even under prolonged periods ofstorage.

The membrane can incorporate an electrochemical activator and asubstrate-specific enzyme, both diffusibly dispersed in a membranedeposited onto an electrode of the sensor where glucose is oxidised. Theterm “electrochemical activator” as used herein refers to any compoundthat is capable of activating the enzyme that transfers electronsbetween glucose and the working (detection) electrode of the sensor. Theelectrochemical activator can be a polymeric redox mediator.Alternatively, monomeric electrochemical activators can also be used,such as water soluble ferrocene derivatives, osmium-bipyridinecomplexes, ruthenium complexes (e.g. penta-amine pyridine ruthenium andRu(NH₃)₆ ³⁺) as well as hexacyanoferrate and hexacyanoruthenate. In someembodiments of the invention, the electrochemical activator containsredox-active metal ions. Examples of such metal ions are silver, gold,copper, nickel, iron, cobalt, osmium or ruthenium ions or mixturesthereof.

In general, suitable polymeric redox mediators to be incorporated intothe nanoparticulate membrane of the invention should have a chemicalstructure which prevents or substantially reduces the diffusional lossof the redox species during the period of time that the sample is beinganalyzed. The diffusional loss of the redox mediator can be reduced byrendering the polymeric redox mediator non-releasable from the workingelectrode in the sensor. This can be achieved by binding or immobilizingthe redox mediator, for example, by covalently attaching orbiconjugation of the redox mediator to a polymer on an electrode.Alternatively, the redox mediator can be immobilized by providing abinder having countercharge species or species having high affinity forthe redox mediator. In one embodiment of the invention, one type ofnon-releasable polymeric redox mediator comprises a redox speciescovalently attached to a polymeric compound. Such redox polymerstypically are transition metal compounds, wherein a redox-activetransition metal-based pendant group is covalently bound to a suitablepolymer backbone, which on its own may or may not be electroactiveitself. Examples of this type are poly(vinyl ferrocene) and poly(vinylferrocene co-acrylamide). Alternatively, the polymeric redox mediatormay comprise an ionically-bound redox species. Typically, thesemediators include a charged polymer coupled to an oppositely chargedredox species. Examples of this type include a negatively chargedpolymer such as Nafion® (Dupont) coupled to a positively charged redoxspecies such as an osmium or ruthenium polypyridyl cation or vice versaa positively charged polymer such as poly(1-vinyl imidazole) coupled toa negatively charged redox species such as ferricyanide or ferrocyanide.Furthermore, the redox species can also be coordinatively bound to thepolymer. For example, the redox mediator may be formed by coordinationof an osmium or cobalt 2,2′-bipyridyl complex to poly(1-vinyl imidazole)or poly (4-vinyl pyridine). Another example is poly(4-vinyl pyridineco-acrylamide) coordinated with an osmium 4,4′-dimethyl-2,2′-bipyridylcomplex. Useful redox mediators as well as methods for their synthesisare described in U.S. Pat. Nos. 5,264,104; 5,356,786; 5,262,035;5,320,725; 6,336,790; 6,551,494; and 6,576,101.

In a further embodiment of the invention, the electrochemical activatoris selected from the novel class of redox polymers that is described indetail later herein. Briefly, this novel class of redox polymerscomprises poly(vinyl ferrocene), poly(vinyl ferrocene)-co-acrylamide,poly(vinyl ferrocene)-co-acrylic acid, and poly(vinylferrocene)-co-acrylamido-(CH₂)_(n)-sulfonic acid, and poly(vinylferrocene)-co-acrylamido-(CH₂)_(n)-phosphonic acid, wherein n is aninteger from 0 to 12, preferably 0 to 8.

The membrane of the invention can also incorporate a redox polymer thatis cross-linked with a protein such as an enzyme or antigen andimmobilised on an electrode surface.

One embodiment of the sensor, comprises a chamber for holding the testsample, whereby the chamber is bounded at least between a working areaon a working electrode and a working area on a reference electrode. Alsoin this embodiment, the oxidoreductase enzyme and water soluble redoxpolymer is coated on the working area of the working electrode.

Referring to the figures, a sensor of the invention is described asfollows. FIG. 1 shows an exploded isometric view of a tip-fillingbiosensor 2 according to an embodiment of the present invention. Thebiosensor 2 includes a stack made up of several layers. The stackincludes, from bottom to top according to FIGS. 1 and 2, a substratelayer 4, a working electrode 6, a spacer 8, a counter electrode 10, anda top layer 12. The spacer 8 spaces apart the working electrode 6 andcounter electrode 10 to thereby electrically insulate them. A recess 14,formed between two legs of a bi-furcated end of the spacer 8, defines asample chamber 14 (see FIG. 2A) between the working electrode 6 and thecounter electrode 10. In this manner, the sample chamber 14 is boundedor defined on the top and bottom by opposite facing surfaces of thecounter electrode 10 and the working electrode 6 respectively; and onthe sides by sidewalls the spacer 8. One side of the sample chamber 14is exposed as shown in FIG. 2. This surface of the working electrode 6is referred to as a working surface. A sensing chemistry materialscarrier, such as a nanocomposite membrane 18, is disposed in the samplechamber 14. This membrane 18 is in physical contact with the workingelectrode 6. The sensing chemistry materials preferably include anelectron transfer agent, such as a diffusible redox mediator (cannot beshown). The redox mediator and other sensing chemistry materials will bedescribed in detail later. A vent hole 16 is formed in the top layer 12and the counter electrode 10 through to the sample chamber 14. Thesubstrate 4 and the top layer 12 are recessed such that portions 20, 22(FIG. 2B) of the working electrode 6 and the counter electrode 10 areleft exposed so that they are connectable to an electronic circuit.

The sample chamber 14 is configured or shaped so that when a sample oranalyte is provided in the chamber 14, the analyte is in electrolyticcontact with both the working electrode 6 and the counter electrode 10.This electrolytic contact allows an electrical current, mediated by theredox mediator, to flow between the electrodes 6, 10 to effectelectrolysis (electrooxidation or electroreduction) of the analyte. Theredox mediator enables electrochemical analysis of molecules of theanalyte which may not be suited for direct electrochemical reaction onthe working electrode 6. The volume of the sample chamber 14 can bebetween 0.1-1 μl, although other volumes are also possible. A region ofthe sample chamber 14, referred to as a measurement zone, contains onlythe portion of the analyte that is interrogated during the analyteassay. In the biosensor 2 in FIG. 1, the measurement zone has a volumethat is approximately equal to the volume of the sample chamber 14.However, it should be noted that a smaller measurement zone, such as 80%or 90% of the size of the sample chamber 14 is also possible.

The height of the sample chamber 14, as defined by the thickness ofspacer 8 is preferably small to promote rapid electrolysis of theanalyte, as more of the analyte will be in contact with surfaces of theelectrodes 6, 10 for a given analyte volume. In addition, a samplechamber 14 of a small height helps to reduce errors from diffusion ofanalyte into a smaller-sized measurement zone from other portions of thesample chamber 14 during the analyte assay, because diffusion time islong relative to the measurement time. Typically, the thickness of thesample chamber is no more than about 0.2 mm. Preferably, the thicknessof the sample chamber is no more than about 0.1 mm and, more preferably,the thickness of the sample chamber is about 0.05 mm or less.

The substrate 4 and the top layer 12 may be formed from an inertnon-conducting material, such as polyester. Alternatively, the substrate4 and the top layer 12 may be formed from a molded carbon fibercomposite. The working electrode 6 preferable has a relatively lowelectrical resistance and is typically electrochemically inert over thepotential range of the biosensor during operation. Suitable materialsfor forming the working electrode 6 include gold, carbon, platinum,ruthenium dioxide, palladium, and conductive epoxies, such as, forexample, ECCOCOAT CT5079-3 Carbon-Filled Conductive Epoxy Coating(available from W.R. Grace Company, Wobum, Mass.), as well as othernon-corroding materials known to those skilled in the art. The counterelectrode 10 may also be formed using any of these materials suitablefor forming the working electrode 6. The working electrode 6 and thecounter electrode 10 may be deposited on the surfaces of the substrate 4and the top layer 12 by any suitable methods, for example by vapordeposition or printing.

The spacer 8 is typically constructed from an inert non-conductingmaterial such as pressure-sensitive adhesive, polyester, Mylar®,Kevlar®, or any other strong, thin polymer film, or, alternatively, athin polymer film such as a Teflon® film, chosen for its chemicalinertness. Other spacers include layers of adhesive and double-sidedadhesive tape (e.g., a carrier film with adhesive on opposing sides ofthe film).

In one specific embodiment, the substrate 4 and the top layer 12 arepolyester films, the working electrode 6 is a screen-printed carbonlayer, the counter electrode 10 is a screen-printed Ag/AgCl layer andthe spacer 8 is a double-sided adhesive tape.

During use of the biosensor 2, the exposed side of the sample chamber 14is used to contact an analyte, such as blood or a serum. The samplechamber 14, with the nanocomposite membrane 18 therein, receives theanalyte for analysis thereof by wicking or capillary action. Dependingon the type of redox mediator, the diffusible redox mediator may diffuserapidly into the analyte or diffusion may occur over a period of time.Similarly, the diffusible redox mediator in the membrane 18 may firstdissolve and then diffuse into the analyte, either rapidly or over aperiod of time. If the redox mediator diffuse over a period of time, auser may be instructed to wait a period of time before measuring theanalyte concentration to allow for diffusion of the redox mediator.

It should not to be construed that the structure and construction of abiosensor is limited to that described above; biosensors having otherstructures and constructed using other processes are also possible. FIG.2 shows one such biosensor 24 according to another embodiment of theinvention. This biosensor 24, which is a variant of the biosensor 2 inFIG. 1, includes the layers 4, 6, 10, 12 and the membrane 18 of thebiosensor 2. However, the spacer 8 in this embodiment includes a firstspacer portion 8A and a second spacer portion 8B separated by a gap 26therebetween. This gap 26 defines a sample chamber 14 when the spacer 8is sandwiched between the working electrode 6 and the counter electrode10. Two opposing sides of the sample chamber 14 are exposed. Therefore,no vent hole is necessary in the biosensor 20.

Some other biosensors are disclosed in U.S. Pat. No. 6,338,790, Feldmanet al., entitled “Small Volume in vitro analyte sensor with diffusibleor non-leachable redox mediator.” Some of these biosensors include morethan one counter electrode 6.

A large variety of oxidoreductases can be employed in a sensor of thepresent invention. One function of the enzymes in a sensor is tocatalyse the oxidation or reduction of the enzyme substrate by theremoval or addition of electrons. For example, where the substrate oranalyte to be detected is glucose, glucose oxidase may be used tooxidize glucose into gluconic acid. Although it may be thermodynamicallyfeasible for the oxidation of glucose to proceed in the absence of anenzyme, the presence of a suitable oxidoreductase helps to acceleratethe oxidation reaction, thereby allowing enzyme activity and substrateanalysis to be easily studied. Besides, such enzymes can be obtainedcheaply and readily.

In some embodiments of the invention, the oxidoreductase is selectedfrom the group consisting of glucose oxidase, hydrogen peroxidase,horseradish peroxidase, xanthine oxidase, cholesterol oxidase, hydrogenhydrogenase, lactate dehydrogenase, glucose dehydrogenase and NADHdehydrogenase, sarcosine oxidase, lactate oxidase, alcoholdehydrogenase, hydroxybutyrate dehydrogenase, glycerol dehydrogenase,sorbitol dehydrogenase, malate dehydrogenase, galactose dehydrogenase,malate oxidase, galactose oxidase, xanthine dehydrogenase, alcoholoxidase, choline oxidase, xanthine oxidase, choline dehydrohenase,pyruvate dehydrogenase, pyruvate oxidase, oxalate oxidase, bilirubinoxidase, glutamate dehydrogenase, glutamate oxidase, amine oxidase,NADPH oxidase, urate oxidase, cytochrome C oxidase, actechol oxidase andmixtures thereof.

Three commonly used conventions for the labeling of groups in thePeriodic Table are, namely, the new IUPAC convention, the old IUPACconvention, (also known as the European convention), as well as the CASgroup labeling convention (also known as the American convention). TheCAS convention is used in the present application. In the CASconvention, Groups IA, IIA, IIIA and IVA refer to the main groupelements of Group 1 (Li, Na, K, etc.), 2 (Be, Mg, Ca, etc.), 3 (B, Al,Ga, etc.) and 4 (C, Si, Ge, etc.), respectively, while Groups IB, IIB,IIIB, IVAB, VB, VIB, VIIB and VIIIB refer to the transition elements.Groups IA, IIA, IIIA and IVA under the CAS convention are equivalent toGroups IA, IIA, IIIB, and IVB, respectively, under the old IUPACconvention, and equivalent to Groups 1, 2, 13, and 14, respectively,under the new IUPAC convention. Groups IB, IIB, IIIB, IVAB, VB, VIB,VIIB and VIIIB under the CAS convention are equivalent to Groups IA,IIA, IIIA, IVA, VA, VIA, VIIA and VIIIA, respectively, under the oldIUPAC convention, and equivalent to Groups 3, 4, 5, 6, 7, 8-10, 11, and12, respectively, under the new IUPAC convention.

Nanoparticles that can be used can be any inorganic oxide of an elementselected from Group IA, IIA, IIIA, IVA, IB, IIB, IIIB, IVAB, VB, VIB,VIIB or VIIIB of the Periodic Table. The nanoparticle may have anysuitable dimension and shape, as long as they are able to provide anefficient diffusional pathway for the glucose molecules to betransported into the membrane to a location near to the surface of theoxidizing electrode. Furthermore, nanoparticles used in the inventioncan be porous or non-porous. The average size of the inorganicnanoparticles used in the membrane of the invention typically rangesfrom about 5 nm to about 1 μm, or from about 100 to about 1000 nm,including from about 100 to about 500 nanometers, or about 200 to about300 nanometers. The size of nanoparticles can be selected according tothe intended application (for example, to influence the length of thediffusional pathway as mentioned above), or also to alter the viscosityor density of the slurry ink that is used for the preparation of themembrane of the invention.

Examples of oxides that are suitable for use in the membrane of theinvention include, but are not limited to, lithium manganese oxide,magnesium oxide, zinc oxide, cobalt oxide, yttrium oxide, niobium oxide,calcium oxide, lanthanum oxide, cerium oxide, aluminium oxide, silicondioxide and mixtures thereof.

In some embodiments, the membrane incorporates nanoparticles ofaluminium, silicon, magnesium or zinc oxide(s). The present inventorsfound that incorporation of such oxides e.g. alumina or silica into themembrane facilitates the preparation of membranes as well as impart goodmechanical strength to the membrane, such that it does not crack, evenafter long periods of use.

With respect to silica particles, any suitable kind of silica particles(for example, fumed silica or colloidal silica) can be used in theinvention. Silica particles as every other particles of an inorganicoxide as defined herein can be selected based on a variety of factors,such as its diameter, aspect ratio, average pore size, or shape. Theseparameters can be chosen for achieving a desired transportcharacteristic in the nanoparticulate membrane. The choice of suitableparticles can also be dependent on the choice of deposition technique.The silica particles may have a size from 5 to about 000 nanometers(nm), or from about 100 to about 1000 nm, including from about 100 toabout 500 nanometers, or about 200 to about 300 nanometers. Silicaparticles can be synthesized in the laboratory or obtained fromcommercial suppliers. Colloidal silica for example (Chemical AbstractsNumber 7631-86-9) is commercially available from many suppliers. Forexample, it is sold under the trade name Snowtex® from Nissan Chemicalsor under the trade name NYACOL® from Nyacol Nanotechnologies, Inc.

The thickness of the nanoparticulate membrane of the invention rangesfrom 50 to 1000 micrometers (μm), or from 100 to 700 μm, or preferablyfrom 250 to 500 μm. The thickness of the membrane may depend on severalfactors such as desired electrode size, or the constituents of themembrane, for example. It may be controlled by the choice of thedeposition technique (screen printing, dip coating or spin coating toname a few), the content of the nanoparticulate material or theconcentration of the ink slurry. In case screen printing is used fordeposition of the ink slurry from which the membrane is made, thethickness of the membrane can be controlled inter alia via the mesh sizeof the screen.

In yet another embodiment of the sensor, the nanoparticulate membrane ofthe invention can further comprise a polymeric binder. Any suitablepolymeric binder can be used in the membrane, includingelectrostatically inert polymers, ionic polymers, polymers capable ofacquiring a net charge, polymers capable of providing biconjugation, andproteins. Polyurethane, cellulose or elastomeric polymers are examplesof polymers which are electrostatically inert. Examples of polymers thatare capable of acquiring a net charge, thereby becoming eitherpositively or negatively charged, are nitrogen-containing heterocyclessuch as pyridine or imidazole. Glycoproteins are a class of proteinsuseful in the present application. Specific examples include avidin,biotin and streptavidin, which can conjugate with electrochemicalactivators present in the membrane to form a suitable polymeric binderfor use in the present invention. Useful binders as well as methods fortheir synthesis are described in U.S. Pat. No. 6,592,745, for example.

In one embodiment, the polymeric binder is a polymer or copolymercomprising monomer units selected from the group consisting of vinylpyridine, vinyl imidazole, acrylamide, acrylonitrile and acrylhydrazideand acrylic acid monomer units. A specific example of a polymeric binderhaving monomers selected the group is vinyl pyridine. A binder derivedfrom these monomeric units are suited for glucose sensing applicationsinvolving blood samples for example because of its dual function ofbinding and analyte regulating in the membrane. By incorporating abinder such as vinyl-pyridine into the membrane, the membrane does notbreak up on hydration, but swells to form a gelled layer holding up thevarious components of the membrane on the screen-printed carbon surface.Mediators, enzymes and analytes such as glucose can then move freelywithin this layer, whereas interfering species, such as red blood cellscontaining oxygenated hemoglobin are excluded from entering the membranedue to electro static repulsion. Anionic ascorbic acid and uric acid areexpelled by the anionic binder, and the partition of dissolved oxygeninto the nanoparticulate membrane is largely minimized owing to thehighly hydrophilic nature of this layer.

The invention is also directed to a process for producing anon-conductive, nanoparticulate membrane. The preparation of ananocomposite, slurry ink comprising an electrochemical activitator suchas a redox polymer, an enzyme and nanoparticles can be carried out in acommercially available mixer, blender or stirrer, depending on thequantity, viscosity and homogeneity of the slurry. The slurry can beprepared in any suitable liquid or dispersion medium, for example, polarsolvents, aqueous solutions, PBS buffer, and organic compounds orsolvents (e.g. alcohol) which can facilitate the processing of thenanoparticulate membrane.

Suitable proportions of components which are used to form the slurry inkvary. For example, the composition may be varied according to theapplication, or on the enzyme used, or the deposition technique that isemployed for depositing the slurry ink and the amounts used for eachcomponent can be determined empirically. For example a suitablecomposition for a slurry ink that is used in the manufacture of amembrane of a glucose sensor may comprise the components in thefollowing range—glucose oxidase: 0.10-1.0 mg/ml; redox mediator: 5-50mg/ml; nanoparticles: 10-200 mg/ml; binder: 10-300 mg/ml. In thisexample, no standing period is required, but the slurry ink can be usedimmediately. However, for other preparation the slurry ink may have tostand for a suitable period of time before being applied onto a suitablesubstrate. In another specific example, the slurry ink may compriseglucose oxidase, poly(VFc-co-AA), alumina nanoparticles and PVPACbinder, mixed with a mixing ratio ranging from 1:50:150:200 to1:40:200:300 weight parts. A redox polymer of the invention is added tothe mixer where it is homogenised with the enzyme, nanoparticles andwater. Thereafter, the homogenized mixture is applied on any suitablesubstrate. A variety of deposition techniques can be employed to depositthe membrane onto a surface, including, but not limited to, spraying,painting, dip coating, spin coating, inkjet printing and screenprinting.

As mentioned above, the concentration of a redox mediator in the slurryink may vary, for example, from 5 to 50 mg/ml. In one example describedbelow where a vinylferrocene-co-acrylamide redox mediator was employed,10 to 20 mg/ml of the mediator was added to the slurry ink. In oneembodiment, the concentration of the redox mediator in the nanocompositeink is about 15 mg/ml.

The concentration of enzyme in the slurry ink may also vary. A typicalrange is from 0.1 to 1 mg/ml. In one embodiment, the concentration ofenzyme in the nanocomposite ink is about 0.2 mg/ml.

The formulation of the nanocomposite ink can be varied as follows. Sincethe catalytic reaction occurs between the mediator and enzyme, theconcentration of the mediator must be high enough to have a highsensitivity and linear relationship between the catalytic oxidationcurrent and glucose concentration. If the amount of mediator is limited,the amperometric response of the sensor will plateau off even withincreasing glucose concentrations in the sample. When this happens, theamount of mediator becomes a bottleneck, and the sensor reading becomesinstead dependent on the amount of mediator in the membrane, rather thanglucose concentration in the sample. For the specific examples describedlater, it was found that the optimized mediator concentration was about15 mg/ml and the optimal concentration of GOX was about 0.20 mg/ml.

The invention also relates to new ferrocene based redox polymers thatare amongst other uses well suited for being used as electrochemicalactivator in glucose sensors of the invention as well as in any as wellas any other known electrochemical detection of analytes, for example.Although ferrocene-containing monomers usually undergo free radicalpolymerization with great difficulty, the inventors have found thatredox polymers containing ferrocene can be elegantly and readilyprepared using an alcoholic medium prepared from, for instance, amixture of ethanol and water, together with a persulfate salt as radicalinitiator.

Whilst any organometallic redox species can be used as a redox mediator(e.g. nickelocene and cobaltocene), ferrocene-based redox mediators arepreferred, for example due to the suitable redox potential derived fromthe oxidation of ferrocene to ferrocinium ion.

Ferrocene derivatives can be used as diffusional electron transfermediators in homogeneous systems. It should be noted that diffusionalmediators are typically low in molecular weight and can leach out of theelectrode and be lost in the sample that is being measured. For thisreason, sensors based on diffusional mediators are suitable asdisposable sensors which are used once and disposed immediatelythereafter.

Ferrocene derivatives can also be used as mediators that are immobilisedon an electrode surface and then attached to a protein molecule, such asan enzyme or an antigen, via crosslinking between crosslinkablefunctional groups found both in the enzyme and in a side chain of theredox polymer.

Suitable polymerisable ferrocene derivatives that can be used as a firstmonomer to form a redox polymer should possess a side chain unit havingan unsaturated bond, such as a C—C double or triple bond, or a N—Ndouble bond or a S—S double bond. Examples of such side chain unitsinclude alkenyl groups, represented by the general formula R₁—C═C—. Thedouble bond can be located at any position along the carbon chain.Aromatic groups e.g. phenyl, toluoyl and naphthyl groups can also beused. Furthermore, the polymerisable group can also comprise substitutedC-atoms wherein a halogen (e.g. fluorine, chlorine, bromine or iodine),oxygen or hydroxyl moiety for example, substitutes one or more hydrogenatoms on carbon atoms in the group. Further examples include an alkynyland a disulphide group.

In a preferred embodiment, the polymerisable ferrocene derivative isselected from the group consisting of vinyl-ferrocene,acetylene-ferrocene, styrene-ferrocene and ethylene oxide-ferrocene.

The presence of an unsaturated bond in these derivatives would allow theferrocene molecule to be attached to a polymer backbone viacopolymerisation with another species having also at least oneunsaturated C—C double or triple bond, or a N—N double bond or a S—Sdouble bond, via free radical polymerisation.

For the second monomer unit that is used in copolymerisation with thepolymerisable ferrocene derivative, any suitable acrylic acid derivativehaving a primary acid or base functional group capable of acquiring anet charge can be used. This means that the invention provides forpositively as well as negatively charged polymers and thus ensure thatconducting bilayers as explained above can be formed, irrespective ofthe net charge of the complex formed between capture molecules andanalyte molecules. In general, there are two requirements for selectinga suitable acrylic acid derivative for use as a monomer. In order for itto copolymerise with the ferrocene derivative, it should possess of atleast one unsaturated bond, which can be provided by a C—C double ortriple bond, or a N—N double bond or a S—S double bond for example.Secondly, the acrylic acid derivative should be able to function as aBronsted-Lowry acid or base by producing H⁺ ions or by accepting H⁺ions, respectively. Examples of functional groups which can provide aBronsted-Lowry acid or base function include primary amine groups whichcan accept H⁺ ions to form charged amine groups, or carboxyl groups, orsulfate which can donate H⁺ ions when the acid functionalitiesdissociates to release H⁺ ions. In this respect, it is noted thatalthough the use of primary amine groups is preferred in the presentapplication, it is evident for the skilled person that also secondary ortertiary amine groups present in the acrylic acid derivative can be usedin order to generate a positively charged redox polymer. In thisrespect, it is also noted that the acid or base functionality, althoughit is a primary one, does not need to be a terminal group, but in caseof a branched side chain can be present “within” the shorter one of theside chains.

While any suitable acrylic acid derivative having an acid or basefunctional group can be used, preferred monomers that are used as thesecond monomer in a redox polymer of the present sensor is an acrylicacid derivative represented by the general formula (I):

wherein R is selected from the group consisting of C_(n)H_(2n)—NH₂,C_(n)H_(2n)—COOH, NH—C_(n)H_(2n)—SO₃H, and NH—C_(n)H_(2n)—PO₃H, whereinthe alkyl chain is optionally substituted, and wherein n is an integerfrom 0 to 12, preferably 0 to 8. The alkyl group can thus be straightchained or branched, and can also comprise double or triple bonds or acyclic structure such as cyclohexyl. Examples of suitable aliphaticmoieties within the substituent R are methyl, ethyl, propyl, isopropyl,butyl, isobutyl, pentyl, isopentyl, hexyl, cyclohexyl, or octyl to namea few. The aliphatic group can further be substituted by an aromaticgroup such as phenyl, a halogen atom, a further base or acid group, oran O-alkyl group, for example. Exemplary aromatic groups that can bepresent as substituents are phenyl, toluoyl or naphthyl. The halogenatom can be selected from fluoride, chloride or bromide. Examples ofsuitable o-alkyl groups are methoxy, ethoxy, propoxy or butoxy, whereasthe n-alkyl group is selected from —NHMe, —N(Me)₂, —N(Ethyl)₂ or—N(Propyl)₂.

The monomer of the acrylic acid derivative, if not commerciallyavailable, can be made starting via nucleophilic substitution from acrylamide, for example, by reaction of its terminal NH₂-group with ansuitable activated derivative of an acid or base compound having analkyl chain as defined here. For example, acryl amid may be reacted with4-bromobutanoic acid or a ester derivate thereof, yielding therespective acryl amid monomer. An analogous procedure can be used forthe sulfonic or phosphoric acid derivatives.

Typically, for biological samples, the pH can be between about 6.5 to7.5. Under such a pH range, acrylamide units in the redox polymer canacquire a positive charge i.e. become cationic. This positive chargebrings the ferrocene moieties in the polymer, via electrostaticinteraction, to a much closer proximity to the redox centers on glucoseoxidase (where glucose oxidase is the selected oxidoreductase enzyme)because glucose oxidase is negatively charged, i.e. anionic, in this pHrange.

In one sensor of the present invention, the oxidoreductase enzyme iscovalently linked to the redox polymer by cross-linkages. The redoxpolymers of the invention can be co-immobilized with the oxidoreductaseenzyme at an electrode surface, making the enzyme anintegral(functional) part of the electrode. Coimmobilisation of enzymeand mediator can be achieved by labeling the enzyme with the redoxmediator, followed by enzyme immobilization on the electrode surface.Alternatively, the redox polymer can be immobilized on the electrodesurface first, and then the enzyme is immobilized in the redox polymer.It is also possible to immobilize both enzyme and redox polymer in amatrix formed from a conducting polymer.

In another sensor of the present invention, the oxidoreductase enzymeand redox polymer are diffusibly dispersed in a nanoparticulate membranecomprising nanoparticles of at least one inorganic oxide of an elementselected from Group IA, IIA, IIIA, IVA, IB, IIB, IIIB, IVAB, VB, VIB,VIIB or VIIIB of the Periodic Table.

In such a sensor, the redox polymers operate as a diffusional mediatorsshuttling between electrode surfaces and the test sample. Ananoparticulate membrane which incorporates a redox polymer not onlyprovides electron mediating function, but also provides analytefiltering function to prevent electrodes from coming into contact withother electrochemically active materials in the sample. Thenanoparticles in the membrane provide microchannels in which analytemolecules can diffuse into in order to reach the oxidizing electrode inthe sensor.

In an embodiment of a sensor of the invention, the redox polymers offormula (I) have a molecular weight of between about 1000 and 5000Daltons, or preferably between about 2000 and 4000 Daltons.

In another embodiment of the invention, the redox polymer of formula (I)has ferrocene loading between about 2% to 17%, or 3% and 14%. Highlevels of ferrocene loading, preferably at least above 3%, aredesirable. Usually, a low level of ferrocene loading would impose alimit to the glucose concentration that can be measured. For example,where glucose concentration is much higher than the mediating capacityof the ferrocene molecules present, the amperometric response that isgenerated may be limited by the small number of mediating ferrocenemolecules, resulting in an inaccurate measurement. Therefore, byemploying the redox polymers of formula (I) having a higher level offerrocene loading, the upper limit of glucose concentrations that can betested with the sensor is raised and thus smaller volumes of samplerequired.

The present invention is also directed to a process for preparing awater soluble, redox polymer. The process essentially involvespolymerising a first monomer unit of a polymerisable ferrocenederivative with a second monomer unit comprising an acrylic acidderivative, such as a primary, secondary or tertiary acrylamide, toproduce a copolymer. The acrylic acid derivative possesses an acid orbase functional group capable of acquiring a net charge. Importantly,the polymerization reaction is carried out in an aqueous alcoholicmedium in the presence of an initiator.

The addition sequence of the monomers and initiator can be varied. Forexample, it is possible to mix the first and second monomer in alcoholicmedium, and then add the initiator to the initiate the reaction. It isalso possible to dissolve one of the monomers in aqueous alcoholicmedium first, and then add the initiator to it, before adding the othermonomer to the mixture.

An alcoholic medium can be prepared with any organic alcohol, forexample, aliphatic alcohols such as ethanol, or aromatic alcohols suchas phenols. The volumetric ratio is usually within the range of ca 5:1to 1:1 (alcohol/water). In some embodiments, it is about 3:1.

In an embodiment of the process according to the invention,polymerisation is carried out using an aqueous alcoholic solventcomprising ethanol and water in a volumetric ratio of between about 2:1and 3:1.

Although polymerisation may proceed without the addition of aninitiator, it is desirable to add a radical initiator which attacks theelectron-rich centres found at the unsaturated bonds in the monomers.Accordingly, in another embodiment of the invention, polymerization isinitiated by adding a free radical initiator.

Any free radical initiator can be used. Examples include inorganic saltssuch as persulfate salts, as well as organic compounds such as benzoylperoxide or 2,2′-azo-bis-isobutyryinitrile (AIBN), which are able toproduce radical fragments called initiator fragments, each of which hasone unpaired electron which can function as a free radical which attackthe unsaturated bonds in the monomer units.

In some embodiments, the free radical initiator is selected form thegroup consisting of ammonium persulfate, potassium persulfate and sodiumpersulfate.

In one embodiment of the invention, the weight ratio of free radicalinitiator added is between about 20 mg to 40 mg per 1 gram of monomer.The inventors have found that the amount of radical initiator affectedthe degree of polymerization. High amounts of radical initiatorsignificantly reduced polymerization efficiency, resulting in redoxpolymers having lower molecular weight. This also meant that relativelylittle radical initiator was needed in the polymerization process,compared to normal free radical polymerization reactions. Apart from thequantity of free radical initiator used, the addition sequence ofreactants (see below in relation to a process of the invention), alsoaffected polymerization efficiency.

The process according to the invention can be carried out at standardconditions of room temperature and pressure. However, in order toaccelerate the reaction, it is generally preferred to carry out thereaction mixture under reflux. Care must also be taken not to use anexcessively high temperature which might lead to the decomposition ofthe polymer or the reactants. Thus, a suitable upper limit is generallybelow 100° C. In a preferred embodiment of the process, polymerizationis carried out under reflux at a temperature of between about 60° C. to80° C.

In a further embodiment, polymerisation is carried out under reflux inan inert atmosphere. An inert atmosphere can be provided by nitrogengas, or helium gas or argon gas for instance.

The length of time that is required for polymerization can be dependentupon the temperature used and the amount of initiator added to thereaction broth. Typically, polymerization is carried out for a period oftime between 10 to 40 hours, and preferably for about 24 hours.

One embodiment of the inventive process further comprises a producing apre-reaction mixture prior to polymerizing said first and secondmonomers, comprising:

-   -   dissolving the acrylic acid derivative monomer unit in an        aqueous alcoholic medium, then    -   adding the free radical initiator, and then    -   adding the polymerisable ferrocene derivative monomer unit to        the mixture.

In a further embodiment of the above process, the feeding ratio ofacrylic acid derivative to polymerisable ferrocene derivative in thepre-reaction mixture that falls between about 5% and 15% of the weightof monomer added is preferable in order to obtain a redox polymer havinga suitable molecular weight and viscosity.

In yet a further embodiment, the polymerisable ferrocene derivativemonomer unit is dissolved in an aqueous alcoholic medium prior to beingadded to the reaction mixture.

In an embodiment of the process of the invention, the redox polymer isprecipitated in an organic solvent. Organic solvents that can be used todissolve monomers involved in polymerization include ether, ketone andalcohol for example.

EXAMPLES Example 1 Construction of a Diffusional Mediator Biosensor

FIG. 1 shows an exploded isometric view of a tip-filling biosensor 2according to an embodiment of the present invention. The manufacturingprocess of the biosensor 2 according to the specific embodiment is nextdescribed. Firstly, an array of carbon electrodes, such as of Electrodag423SS available from Acheson Colloids Co., Ontario, Calif., U.S.A., isprinted using a suitable mask on a polyester-film substrate. The printedsubstrate is then dried at a temperature of around 70° C. for apredetermined period of time, for example twenty-four hours. Thereafter,a double sided tape with holes appropriately formed therein is placed onthe printed substrate. These holes would eventually define the recesses14 and the openings for exposing the electrode portions 20, 22. Auniformed nanoparticulate membrane is then screen-printed, using asuitable mask, on the working surfaces of the carbon electrodes with anaqueous slurry “ink” of PVFcAA (prepared as described in Example 2,below), GOX, a poly(vinylpyridine-co-acrylic acid) (PVPAC) binder andalumina nanoparticles. The resultant structure is then dried at atemperature of around 37° C. in a controlled environment. The thicknessof the nanoparticulate membrane may generally be controlled by adjustingthe total content in the ink while keeping a constant volume applied onthe working area, and is manipulated for example by adjusting the meshsize of a screen in a screen printer or by adjusting the content ofnanoparticulate materials, or the ‘concentration’ of the printingslurry.

While the above-described structure is being formed, an array of Ag/AgClor carbon counter electrodes is similarly screen-printed on a secondpolyester film using a suitable mask and dried. This second polyesterfilm is then positioned over the adhesive tape such that the counterelectrodes are aligned with their corresponding working electrodes.Thereafter, the structure is singulated to produce multiple biosensor 2,one of which is shown in FIG. 1.

Example 2 Synthesis of Poly(vinylferrocene-co-acrylamide),Poly(vinylferrocene-co-acrylic Acid) andPoly(vinylferrocene-co-acrylamido-sulphonic Acid) Copolymers

Glucose oxidase (GOx, EC 1.1.3.4, from Aspergillus niger, 191 units/mg)was purchased from Fluka (CH-9470 Buchs, Switzerland). Ferrocene (Fc),Vinylferrocene (VFc), acrylamide (M), acrylic acid (AC),2-acrylamido-2-methyl-1-propane-sulfonic acid (cat. no. 28,273,“acrylamido-sulfonic acid” or AAS) and persulfate salts were purchasedfrom Sigma-Aldrich (St. Luis, Mo., USA.). All other chemicals such asacetone, ethanol, and phosphate buffered saline used were of certifiedanalytical grade. All solutions that were used were prepared withdeionized water.

UV spectra of polymers produced in the experiment was performed andrecorded on an Agilent 8453 UV-visible spectrophotometer. Molecularweights were determined with a Toyo Soda high performance gel permeationchromatography in water and standard poly(ethylene oxide) andpoly(ethylene glycol) for calibration.

i) Synthesis of Poly(vinylferrocene-co-acrylamide) Polymers

Three samples containing 1.0 g acrylamide dissolved in 10 ml of mixturesolvent of ethanol/water (3 parts to 1 part) were prepared. A 0.30 mlaliquot of 0.10 g/ml oxygen-free persulfate solution was added to eachsample after being deoxygenated for 10 minutes. Three amounts ofvinylferrocene ranging from 0.05 g to 0.16 g were dissolved in degassedethanol to form three vinylferrocene solution samples, the amount offerrocene that is added for each sample being calculated to obtainacrylamide-to-vinylferrocene feeding ratios (w/w) of 95:5, 90:10 and85:15, respectively. Each vinylferrocene sample was then added to anacrylamide-initiator mixture. Reaction mixtures were refluxed at 70° C.for 24 hours in nitrogen atmosphere. After cooling, the reactionmixtures were, separately, added drop-wisely to rapidly stirred acetonein order to precipitate a redox polymer. The precipitated redox polymerwas washed with acetone and purified by multiple water-dissolvingacetone-precipitating cycles. The purified product was then dried undervacuum at 50° C.

ii) Synthesis of Poly(vinyl Ferrocene-co-acrylic Acid) Polymers

Three samples containing 1.0 g acrylic acid dissolved in 10 ml ofmixture solvent of ethanol/water (3 parts to 1 part) were prepared. A0.30 ml aliquot of 0.10 g/ml oxygen-free persulfate solution was addedto each sample after being deoxygenated for 10 minutes. Three amounts ofvinylferrocene ranging from 0.05 g to 0.16 g were dissolved in degassedethanol to form three vinylferrocene solution samples, the amount ofvinylferrocene that is added for each sample being calculated to obtainacrylamide-to-vinylferrocene feeding ratios (w/w) of 95:5, 90:10 and85:15, respectively. Each vinylferrocene sample was then added to anacrylamide-initiator mixture. Reaction mixtures were refluxed at 70° C.for 24 hours in nitrogen atmosphere. After cooling, the reactionmixtures were, separately, added drop-wisely to rapidly stirred acetonein order to precipitate a redox polymer. The precipitated redox polymerwas washed with acetone and purified by multiple water-dissolvingacetone-precipitating cycles. The purified product was then dried undervacuum at 50° C.

iii) Preparation of Poly(vinyl Ferrocene-co-acrylamido-sulphonic Acid)Polymers

Three samples containing 1.0 g acrylic acid dissolved in 10 ml ofmixture solvent of ethanol/water (3 parts to 1 part) were prepared. A0.30 ml aliquot of 0.10 g/ml oxygen-free persulfate solution was addedto each sample after being deoxygenated for 10 minutes. Three amounts ofvinylferrocene ranging from 0.05 g to 0.16 g were dissolved in degassedethanol to form three vinylferrocene solution samples, the amount ofvinylferrocene that is added for each sample being calculated to obtainacrylamide-to-vinylferrocene feeding ratios (w/w) of 95:5, 90:10 and85:15, respectively. Each vinylferrocene sample was then added to anacrylamide-initiator mixture. Reaction mixtures were refluxed at 70° C.for 24 hours in nitrogen atmosphere. After cooling, the reactionmixtures were, separately, added drop-wisely to rapidly stirred acetonein order to precipitate a redox polymer. The precipitated redox polymerwas washed with acetone and purified by multiple water-dissolvingacetone-precipitating cycles. The purified product was then dried undervacuum at 50° C.

Results and Discussion

Co-polymerization of vinylferrocene with acrylamide and its derivativeswere carried out based on conventional radical polymerization reaction.The general reaction equation is depicted in FIG. 6.

However, in order to successfully co-polymerize the monomers, greatattention was given to the terminating effect of vinylferrocene in thesystem. As mentioned in the introduction section, vinylferrocene usuallyacts as radical scavenger in the co-polymerization system. It was foundthat the amount of radical initiator is substantially less that theseneeded in normal polymerization systems. Higher amounts of radicalinitiator significantly reduced polymerization efficiency and themolecular weight of the product. Besides, the addition sequence alsoaffects the polymerization efficiency.

Less than 20% of polymerization was observed when adding the persulfateradical initiator to the solution of vinylferrocene and acrylamide. Thisis probably because the formation of ferrocenium in the reaction mixturewhich resulted in the retardation of polymerization rate and much earlytermination of the polymer chain growth process. As shown in Table 1,under optimal conditions, relative high yields were obtained. TABLE 1Co-polymerization of vinylferrocene, acrylamide, and its derivativesFeeding ratio VFc content Molecular (w/w) Yield (%) (%) weight AA/VFc 804% 3600 95:5 AA/VFc 72 9% 3100 90:10 AA/VFc 56 11% 2400 85:15 AC/VFc 753% 2800 95:5 AC/VFc 55 7% 2500 90:10 AC/VFc 45 6% 2000 85:15 AAS/VFc 856% 4000 95:5 AAS/VFc 75 9% 3500 90:10 AAS/VFc 62 14% 3000 85:15

However, the polymer yields decreased with increasing vinylferrocenefeeding ratio, which indicated that the terminating effect in radicalpolymerization still exists, even though great care has already beentaken in the polymerization process. It was also found that minuteyields were obtained once the reaction mixture became blue, which wasdue to the formation of considerable amount of ferrocenium in thepolymerization solution. Ferrocene loading varied from 3 to 14%, whichis always less than ferrocene content in the monomer feedings.

Ferrocene loading in the redox polymer was determined from elementalanalysis. Energy Dispersive X-ray Analysis (EDX) was used for thispurpose. The energy of electron beam used on samples of the redoxproduced is 120 keV. The X-rays generated by the sample was subject toanalysis by a lithium drifted silicon detector.

The molecular weight of the redox polymer was determined by gelpermeation chromatography. Generally, the redox polymers prepared withhigher ferrocene feeding ratio had lower molecular weight and broadermolecular weight distribution.

Characterization of the Synthesized Redox Polymers

The synthesized copolymers were light-yellow colored, powdery materials.Molecular weights of the copolymers are in between 2000 and 4000Daltons. Fr-IR experiments (see FIG. 7) clearly showed the completedisappearance of vinyl absorption at 1650 suggesting that bothacrylamide and vinylferrocene were successfully polymerized and theresulting redox polymer is of high purity, free of monomers. Furtherevidence can be found in the 1000-1300 cm⁻¹ region. Extremely strongadsorption accompanying by a weak one at 1126 cm⁻¹ indicates thepresence of ferrocenyl units in the redox polymer and the strongabsorption at 1218 cm⁻¹ suggested amide groups in the polymer. UVexperiments, again, confirmed the successful co-polymerization ofvinylferrocene and acrylamide. The minute shoulder at 300 nm is a clearindicative of ferrocene moiety in the co-polymer (see FIG. 8). Havingferrocenyl and amine or carboxylic acid moieties in the redox polymerrendered them with dual-function: redox activity for electron-mediatingand chemical activity for cross-linking with proteins.

Increasing the feeding ratio of vinylferrocene was intended to increasethe proportion of ferrocenyl moiety within the redox polymer. However,varying the amounts of vinylferrocene also affected the polymer yield.The highest yield obtained was when the vinylferrocene feeding ratio wasthe lowest, which is in good agreement with the unusual behavior offerrocenyl compounds in radical polymerization. As indicated in Table 1,although the content of ferrocenyl moiety in the polymer increased withincreasing vinylferrocene feeding ratio, but it is by far not linear atall. It was found that, for biosensing purpose, a vinylferrocene feedingratio of 10% is sufficient, which gives good mediating function and goodeconomy. The amount of initiator used in the polymerization alsoaffected composition and yield of the redox polymer. It was found thatgood redox polymers were obtained when the initiator is in the range of20-40 mg per gram of monomers.

Example 3 Obtaining Cyclic Voltammograms of the Redox Polymers

Redox polymers were prepared in phosphate-buffered saline (PBS)solutions in the presence of 0.0 μg GOx, 10 μg GOx, and 10 μg GOx and 10mM glucose.

Electrochemical tests were performed with an AutoLabpotentiostat/galvanostat running under the general purposeelectrochemical system (GPES) manager version 4.9. A 3-electrode systemcell, housed in a Faraday cage. The electrodes were a (Ag/AgCl)reference electrode, a platinum wire counter electrode and an Au workingelectrode (surface area of 7.94 mm²).

In contrast to vinylferrocene, the redox polymers that were synthesizedhave high solubility in water but are insoluble in most organicsolvents. This characteristic renders the redox polymers ideal for usesas mediators in biosensing, particularly in enzyme-linked biosensingsince most enzymes only work in aqueous media.

FIG. 9 shows typical cyclic voltammograms of the In PBS containing onlythe redox polymers, the voltammograms exhibited highly reversiblesolution electrochemistry: the redox waves centered at ˜0.18 V (vs.Ag/AgCl), the voltammogram has diffusion-limited shape, the magnitude ofthe anodic and cathodic peak current is the same, the peak-to-peakpotential separation is 60 mV, very close to the theoretical value of 59mV at 25° C. These redox waves can be assigned to the oxidation andreduction of ferrocenyl moieties in the redox polymers, which indicateexcellent redox activity of the polymer. The voltammetric experiments,again, demonstrated that vinylferrocene was successfully co-polymerizedwith acrylamide and its derivatives and the ferrocenyl moieties in thepolymers retain their electroactivities. The redox polymers in PBS arein real solution form with free diffusional behavior. Spiking thissolution with varies amounts of glucose did not change the voltammogramat all, which suggests that there is no catalytic oxidation of glucoseby the redox polymers alone. Furthermore, no obvious changes wereobserved when adding small amounts of GOx in the redox polymer solution.The electrochemistry of the resulting solution was practically the sameas the redox polymer alone solution. However, when 10 mM glucose wasadded to this solution, the enzymatic oxidation of glucose by GOxproceeds in the solution. The redox centers in GOx, FAD were convertedto FADH₂. When the electrode potential was scanned past the redoxpotential of the redox polymer, significant amount of ferrocene moietiesin the redox polymer was oxidized to ferrocenium near the electrodesurface. The redox potential of FAD/FADH₂ in GOx is −0.36 V (vs.Ag/AgCl), which is much lower than the ferrocene/ferrocenium couple, theferrocenium moieties in the vicinity of FADH₂ oxidize it back to FAD,and the ferrocenium moieties in the redox polymer are reduced to theoriginal ferrocene moieties. These two reactions form a catalytic cycle,as illustrated in FIG. 4, or in other words, glucose oxidation by GOx ismediated by the redox polymer.

Thus, the catalytic reaction by the redox polymer greatly enhances theoxidation current in the solution containing glucose, as seen in FIG. 9(light grey traces). If the electron-exchange among FADH₂, redox polymerand electrode are all very fast, large amount of ferrocenium moietiesare produced during electrochemical oxidation, and they are, in turn,rapidly consumed by FADH₂. This is the reason for the much lowerreduction current of ferrocenium moieties, as compared to that obtainedin the glucose-free solution. These data suggests that the redoxpolymers function effectively as redox mediators in enzymatic reactions,shuttling electrons from the redox centers of enzyme to electrodesurface.

Example 4 Synthesis of a Membrane Comprising VinylFerrocene-co-acrylamide Cross-Linked with Glucose Oxidase-Bovine SerumAlbumin (GOx-BSA)

The cross-linking reaction of the redox polymer with proteins wascarried out to study the electrochemical properties of the resultingmembrane. The enzyme GOx was used in the present example. Glutaradehydeand poly (ethylene glycol) diglycidyl ether (PEG) were chosen ascross-linkers. Biological grade glutaraldehyde (50% in water, productcode 00867-1 EA) and poly (ethylene glycol) diglycidyl ether (PEGDE)(product code 03800) was obtained from Sigma-Aldrich.

First, poly(vinylferrocene-co-acrylamide) obtained from Example 1 wasdeposited onto a gold electrode. GOx-BSA was modified with thecrosslinkers to provide GOx-BSA with an aliphatic carbon chain with aterminal aldehyde functional group which can provide cross linkage withsuitable functional groups on the immobilized mediator. Subsequently,the modified GOx-BSA was deposited and reacted with the immobilizedinitiator. The aldehyde group on the modified GOx-BSA reacted with theamine group on the PAA-VFc to form a covalent crosslinkages. Afterreaction was carried out, the PAA-VFc-GOx-BSA film was allowed to dry.

The crosslinked PAA-VFc-GOx-BSA film on gold electrode was subjected tovoltammetric analysis. Blank PBS was used, and a potential scan rate of50 mV/s was applied.

FIG. 10 shows a cyclic voltammogram of the PEG cross-linked PAA-VFc withGOx and BSA on gold electrode in blank PBS. As illustrated in FIG. 10,the cross-linked film exhibited exactly as expected for a highlyreversible surface immobilized redox couple (A. J. Bard, L. R. Faulkner,Electrochemical Methods, John Wiley & Sons: New York, 2001.) with littlechange after exhaustive washing with water and PBS, and after numerousrepetitive potential cycling between −0.2 V and +0.8V, revealing ahighly stable surface immobilized ferrocenyl film on gold electrode. Atslow scan rates, <100 mV/s, a remarkably symmetrical signal was recordedas expected for a surface confined one-electron redox system exhibitingan ideal Nernstian behavior: The peak current is proportional to thepotential scan rate, the peak-to-peak potential separation is much lessthan 59 mV, as observed in the case of diffusional behavior in solution(see FIG. 9), and the width of the current at half-peak height is around90 mV. Such results ascertain that all of the ferrocenyl redox centersare allowed to reach the electrode surface and proceed to reversibleheterogeneous electron transfer. Upon adding 10 mM of glucose to the PBSsolution, a typical catalytic electrochemical curve was obtained.However, the reduction peak of the redox polymer disappeared (FIG. 10,light grey trace). This meant that the sensing layer was homogenouslymaintained in the reduced state by the transfer of electrons from thereduced GOx to the ferrocenyl moieties. The rapid response and currentdetected indicated excellent mediating function of the redox polymer thehigh current sensitivity (750 nA/mM glucose) of the biosensors.

Based on these positive results, further examples were carried out toinvestigate the performance of biosensors incorporating redox polymersof the present invention as a diffusional mediator co-dispersed withglucose oxidase in a nanoparticulate membrane.

Example 5 Preparation of a Nanoparticulate Membrane Which IncorporatesCo-Dispersed Diffusional Poly(vinylferrocene-co-acrylamide) and GlucoseOxidase

I) Preparation of Poly(vinylferrocene-co-acrylamide) Redox Polymer

D-(+)-glucose and glucose oxidase (GOX, EC 1.1.3.4, from Aspergillusniger, 191 units mg⁻¹) were purchased from Sigma-Aldrich (St Louis, Mo.,USA). Alumina nanoparticles, with particle size ranging from 10 to 1000nm, were synthesized in house as follows. Aluminum nitrate (Al(NO₃)39H₂0, 88.30 g) was dissolved in 471 ml of water, and then added dropwiseto a base solution prepared from 205.9 ml of concentrated ammoniumhydroxide in 411.93 ml of water. The resulting precipitate was stirredand aged at 25° C. overnight, and then centrifuged for supernatantremoval. After washing, drying, and grinding, the precipitate wascalcinated at 700° C. in air for 3 hours. The resulting gamma-aluminananocrystals have a controllable size range between several tens tohundreds of nanometers.

The phosphate-buffer saline solution (PBS) (pH of 7.4) was prepared fromphosphate salts (0.020 M) and sodium chloride (0.15 M). Thepoly(vinylferrocene-co-acrylamide), glucose and GOX solutions wereprepared with the PBS buffer. Glucose stock solution was allowed tomutarotate for at least 24 h before use. All solutions were preparedwith deionized water obtained from Millipore. All other chemicals usedin the present example were of certified analytical grade.

Poly(vinylferrocene-co-acrylamide) (PVFcAA) redox polymer wassynthesized according to the following procedure: 0.15 g vinylferroceneand 1.0 g acrylamide were dissolved in 10 ml of aqueous alcohol (2 partsethanol: 1 part water). To initiate polymerization, a 0.50 ml aliquot of0.10 g/ml oxygen-free ammonium persulfate solution was added to thereaction mixture after 10 minutes of deoxygenating. The mixture wasrefluxed for 24 h under nitrogen. After cooling, the redox polymer wasprecipitated in acetone. Purification was performed by dissolving thecrude product in water and precipitating in an acetone/water mixture.

II) The Synthesis of Nanocomposite Membrane

The nanocomposite membrane was screen-printed onto carbon strip using anaqueous slurry “ink” of PVFcAA, GOX, a poly(vinylpyridine-co-acrylicacid) (PVPAC) binder and alumina nanoparticles. The aqueous slurry inkwas prepared by mixing PVFcAA as prepared above, GOX,poly(vinylpyridine-co-acrylic acid) (PVPAC) orpoly(vinylpyridine-co-acrylamo-sulfonic acid (PVPPAS) binder and aluminananoparticles into water according to the following range ofconcentrations: Glucose oxidase: 0.20-0.50 mg/ml, mediator: 10-20 mg/ml,nanoparticles: 30-100 mg/ml, PVPPAC or PVPPAS Binder: 40-150 mg/ml. Theslurry ink can be stored or immediately used. When it is desired to coata sensor electrode with a membrane layer, the slurry ink is loaded intoa deposition apparatus such as a screen printing machine, and depositedonto the electrode to form the membrane. Prior to assembly into asensor, the membrane is first allowed to dry.

Example 6 Cyclic Voltammetry Analysis of a Glucose Biosensor Using aDiffusional Poly(vinylferrocene-co-acrylamide) Mediator and GlucoseOxidase Co-Dispersed in a Nanoparticulate Membrane

A sensor was assembled with a screen printed carbon working electrodeand a Ag/AgCL reference electrode in the present example, using a slurryink as prepared in Example 5. The performance of the sensor was analysedusing cyclic voltammetry. All electrochemical measurements were carriedout with a model CHI 660A electrochemical workstation (CH Instruments,Austin, USA) at room temperature. Cyclic voltammetric measurements wereperformed using a conventional three-electrode system, consisting of ascreen-printed carbon working electrode, a miniature Ag/AgCl referenceelectrode (Cypress Systems, Lawrence, Kans., USA ) and a platinum wirecounter electrode. To avoid the spreading of the printing ink beyond the2-mm diameter working area, a patterned hydrophobic film was applied tothe carbon electrode. To avoid electrode fouling and possibleconcentration changes in the ink, fresh electrode and ink were used foreach voltammetric test. All glucose measurements were performed in thePBS solution. In experiments where the pH was varied, 1.0 M HCl and 1.0M NaOH solutions were used to adjust the pH of the PBS buffer. Inamperometric experiments, the working electrode was poised at 0.30 V(vs. Ag/AgCl).

Results and Discussion

A typical cyclic voltammogram of the PVFcAA mediator in a plainnanocomposite ink is shown in FIG. 12. The electrode exhibited classicalfeatures of a diffusion-controlled kinetically fast redox couple. Thepeak current increased linearly with the square root of potential scanrate, and the difference between the reduction and oxidation peakpotential remained unchanged at 59 mV for scan rates up to 200 mV/s,showing that charge transfer from the mediator to the electrode israpid. Spiking this ink with glucose did not change the voltammogram atall, which suggests that there is no catalytic oxidation of glucose bythe mediator alone. Furthermore, practically identical voltammograms, asthat shown in FIG. 12 a, were obtained in the presence of differentamounts of GOX, ranging from 0.10 to 20 mg/ml, indicating that theenzyme does not appreciably affect the electrochemistry of the Fc⁺/Fcredox couple in the ink. However, an addition of a very small amount ofglucose to this ink resulted in an enhanced anodic current and adiminished cathodic current (FIG. 12 b). In addition, as can be seen inFIG. 12 b, the voltammogram was lifted up towards the anodic side aroundthe redox potential of the mediator. Such changes are indicative of atypical chemically coupled electrode process (electrocatalysis). Theelectrocatalysis can be described by the following reaction sequence:Glucose+GOX-FAD+2 H⁺→Gluconolactone+GOX-FADH₂  (1)GOX-FADH₂+2 Fc⁺→GOX-FAD+2 Fc+2 H⁺  (2)Fc→Fc⁺ +e ⁻  (3)

Thus, the GOX-FAD is reduced to the GOX-FADH₂ by the glucose penetratingto the membrane (Eq. 1), electrons are transferred from the GOX-FADH₂ tothe Fc⁺ sites (Eq. 2), and the electrons are then transferred throughthe Fc⁺/Fc sites of the polymeric mediator to the electrode surface (Eq.3). The oxidation of ferrocene moieties at the underlying carbonelectrode accounts for the enhanced anodic current seen in FIG. 12 b.

The rate constant k of the catalytic reaction between Fc and GOX can beestimated from the voltammetric data obtained in the presence of a largeexcess of glucose to ensure that the enzyme is completely reduced. Undersuch circumstances, the reaction between GOX and Fc (Eq. 2) ispractically pseudo-first-order. As shown by Nicholson and Shain, andUaudet and co-workers, the limiting current, I_(L), due to the mediatedredox process between Fc and GOX can be described as follows:I_(L)=nFAC_(Fc)(2D_(Fc)kC_(GOX))^(1/2)  (4)where C_(Fc) and C_(GOX) are the concentrations of Fc and GOX,respectively. D_(Fc) is the diffusion coefficient of Fc, and othersymbols have their usual meanings. As anticipated from Eq. 4, thelimiting current was independent of potential scan rate at sufficientlyslow rates, and was proportional to Fc concentration and the square rootof GOX concentration. These observations justified the use of Eq. 4 indetermining the rate constant of the mediated glucose oxidation. Bycombining Eq. 4 and the peak current, i_(p), expression in linear sweepvoltammetry, the I_(L)/i_(p) relationship is obtained as follows:I_(L)/i_(p)=(2kC_(GOD))^(1/2)/[0.4463(nFv/RT)^(1/2)]  (5)which contains experimental parameters that are easy to determine and iswell-suited for the purpose of determining the rate constant since theelectrode process of Fc/Fc⁺ couple is solely controlled by diffusion.The rate constant was found to be about 3.8×10³ l/s mol, estimated fromdata obtained at slow scan rates, <5.0 mV/s, in solutions containing0.50 mM Fc, 60 mM glucose and 0-30 μM GOX. Such a rate constant suggeststhat PVFcAA efficiently mediates the oxidation of GOX and is anexcellent mediator for coupling the enzymatic oxidation of glucose to anelectrode surface. The rate constant obtained in this work issignificantly larger than those of other ferrocene derivative-GOXsystems previously reported. A possible cause could be the presence ofcationic acrylamide units in the redox polymer at pH 7.4, which bringsthe Fc moieties to a much closer proximity of the redox centers of GOXvia electrostatic interaction since GOX is anionic at this pH.

Example 7 Amperometric Response of a Glucose Biosensor Using DiffusionalPoly(vinylferrocene-co-acrylamide) Mediator and Glucose OxidaseCo-Dispersed in a Nanoparticulate Membrane

A sensor was assembled with a screen printed carbon working electrodeand a Ag/AgCL reference electrode in the present example, using a slurryink as prepared in Example 5. A typical amperometric response of glucosein an air-saturated PBS buffer at the biosensor is shown in FIG. 13 a.Amperometric tests demonstrated that the biosensor has a rapid responsetime and high sensitivity to glucose. At 0.30 V, after spiking theglucose concentration, the oxidation current increased and reached themaximum very rapidly, within 5 s, followed by a gradual transient whichmaintains more than 60% of the peak current for a period of 20 s. Nocatalytic oxidation current was observed in a blank PBS buffer underidentical experimental conditions (FIG. 13 b), but the presence of thenanoparticulate membrane did increase the background current and it tooka considerably long time to drop to a minute level.

In order to obtain a satisfactory performance of the biosensor, theformulation of the nanocomposite ink was optimized. Since the catalyticreaction occurs between the mediator and GOX, the concentration of themediator must be high enough to have a high sensitivity and linearrelationship between the catalytic oxidation current and glucoseconcentration. Otherwise, the fraction of mediated glucose oxidationwill be small and dependent on the amount of mediator in the membrane,instead of the glucose concentration. It was found that a mediatorconcentration of 15 mg/ml was best for our purpose and the optimalconcentration of GOX was found to 0.20 mg/ml, taking into considerationof both sensitivity and biosensor economy (FIGS. 14 a and 14 b). Thepoise potential is expected to affect the amperometric response of thebiosensor; it was therefore examined in the range of 0.0 to 0.70 V. Asillustrated in FIG. 13 c, the current sensitivity increased withincreasing poise potential and reached a plateau at 0.30 V. A slightdecrease in sensitivity was observed when the poise potential becamemore positive than 0.50 V, presumably due to an increased backgroundcurrent. Moreover, too high a poise potential compromises the accuracyof glucose measurements owing to complications from both the muchincreased background current and possible direct oxidation of a numberof electroactive species at the underlying electrode. For amperometricmeasurements of glucose, the potential of the biosensor was thereforepoised at 0.30 V.

The dependence of the catalytic oxidation current of glucose on thethickness of the nanoparticulate membrane was also investigated (FIG. 14d). As can be seen in FIG. 14 d, the catalytic oxidation current reachedmaximum for nanoparticulate membranes with thicknesses of 250-500 μm.Insufficient materials in thinner membranes resulted in lowersensitivity and the disappearance of the current peak, On the otherhand, further increase in membrane thickness beyond 500 μm couldinversely affect the membrane permeability for glucose and the oxidationproducts of the GOX-catalyzed reaction. In addition, longer responsetimes were noted for thicker membranes.

Unlike those utilizing surface-immobilized sensing membrane, theutilization of the non-conductive nanoparticulate sensing membraneoffers great advantages over known disposable glucose biosensors interms of selectivity. In the former systems, the sensing membrane ispart of the electrode and is in direct contact with blood samples. Someconstituents in blood, such blood cells, both red and white, proteinsand ascorbic acid may interact with the sensing membrane and compromisethe accuracy of blood sugar measurements. In this work, thenanoparticulate membrane is non-conductive, and therefore structurallyand functionally is not part of the electrode. Catalytic oxidation ofglucose only takes place at the electrode/nanoparticulate membraneinterface. In other words, no electroactive species exchanges electronswith the electrode unless it passes through the nanoparticulate membraneto reach that interface. Thus, the nanoparticulate membrane provides abarrier to the passage of possible interferences of bulky species inblood such as cells and proteins. When this formulation was used toprint the nanoparticulate membrane, the PVPAC binder serves a dualfunction in the sensing membrane: binding and analyte regulating. Onrehydration, the membrane does not break up, but swells to form a gelledlayer on the screen-printed carbon surface. Reactants, such as glucoseand mediators move freely within this layer, whereas interferingspecies, such as red blood cells containing oxygenated hemoglobin areexcluded. Anionic ascorbic acid and uric acid are expelled by theanionic PVAC polymer, and the partition of dissolved oxygen into thenanoparticulate membrane is largely minimized owing to the highlyhydrophilic nature of this layer. This resulted in a sensing membranewhereby the amount of current generated in response to a given glucoseconcentration varied by less than 5.0% over a hematocrit range of 40-60%and in the presence of 0.20 mM ascorbic and 0.10 mM uric acid. Suchdesirable insensitivity towards the interfering constituents in bloodwas also observed in whole blood samples. Furthermore, thenanoparticulate membrane presented an analyte regulating layer forglucose too, significantly slowing down the transport of glucose so thatthe system was not kinetically controlled, thereby extending the lineardomain through the entire physiologically relevant glucose concentrationrange of 40 to 540 mg/dl.

As mentioned earlier, oxygen affects the sensitivity of the glucosebiosensor because glucose oxidation by dissolved oxygen occurssimultaneously as a side-reaction. Initial amperometric tests on thinnanoparticulate membranes employing a hydrophobic polyvinylpyridine(PVP) binder showed that the response was higher in the absence ofoxygen than that with dissolved oxygen. At low glucose concentrations,e.g. 50 mg/dl, the competition with oxygen caused a significant decrease(˜20%) in peak current. Hence, there was a need to suppress the oxygeninterference in the system to achieve a highly selective and accuratebiosensor. Introduction of acrylic acid units into the hydrophobic PVPresulted in a marked improvement of the biosensor performance. Theresulting nanoparticulate membrane was highly hydrophilic, whichimproved the glucose/oxygen permeability ratio and optimized theaccuracy and linearity of the biosensor response. The two amperometricgraphs for 200 mg/dl glucose solutions bubbled with nitrogen (FIG. 15 a)and oxygen (FIG. 15 b) overlaid nicely with a difference of less than 5%in peak current, showing that the biosensor was rather insensitive tothe oxygen content in the samples.

Example 8 Analysis of Characteristics of a Glucose Biosensor Using aDiffusional Poly(vinylferrocene-co-acrylamide) Mediator and GlucoseOxidase Co-Dispersed in a Nanoparticulate Membrane

A sensor was assembled with a screen printed carbon working electrodeand a Ag/AgCL reference electrode in the present example, using a slurryink as prepared in Example 5. The sensitivity was ˜76 nA/mg/dl for thepeak current for glucose concentrations of ≦600 mg/dl. As seen in FIG.16, the catalytic oxidation current was directly proportional to theglucose concentration up to 600 mg/dl, covering the entirephysiologically relevant blood sugar levels. Interestingly, currentsobtained at any point of time after the current peak was also linearlydependent on the glucose concentration (see FIG. 16 b), providingalternative sampling possibilities within the first 20 seconds of theamperometric tests. The precision was estimated from two series of 20repetitive measurements of 40 and 300 mg/dl glucose solutions. Therelative standard deviations were 4.0% and 8.6%, respectively. Thedetection limit, estimated from 3 times the standard deviation ofrepetitive measurements of 5.0 mg/dl glucose under optimal conditions,was found to be 1.8 mg/dl, which is limited by the charging current ofthe biosensor. More importantly, the blood sample volume needed for asingle test was about 0.20 to 0.30 μl, the smallest sample volumeamongst all the disposable glucose biosensors available on market. Thestability tests were carried out at different temperatures. It was shownthat the biosensor maintained 100% of its initial sensitivity for thefirst 180 days of storage at room temperature, lost 10% of its initialsensitivity after 60 min exposure at 50° C. and about 50% of its initialsensitivity after 60 min at 60° C. This may be due to the loss of enzymeactivity in the biosensor. The proposed method was successfully appliedto the determination of glucose in whole blood (Table 2). TABLE 2Results of blood sugar analysis (average of 10 tests) Glucose ReferenceValue Recovery (%) Sample (mg/dl) (mg/dl)* (+50 mg/dl glucose) WholeBlood 1 80 84 96.3 (Healthy person) Whole Blood 2 110 115 99.2 (Healthyperson) Whole Blood 3 105 105 104 (Healthy person) Whole Blood 4 185 17998.5 (Diabetic patient) Whole Blood 5 155 158 97.1 (Diabetic patient)*Obtained with the YSI blood sugar analyzer.

The results obtained were in good agreement with the reference valuesobtained with a yellow springs blood sugar analyzer (YSI Model 2300).The recoveries obtained were also good enough for practical use.

A series of water-soluble and cross-linkable ferrocenyl redox polymershave been prepared by conventional radical polymerization ofvinylferrocene and acrylamide and its derivatives. The resulting redoxpolymers produced a typical catalytic oxidation current for glucose inthe presence of GOx. The experimental results showed that the redoxpolymers retained their fast electron transfer properties and the GOxretained its catalytic activity after they were introduced in PBS. Theredox polymers having amine or carboxylic acid moiety as one of the sidechains allow them to be conveniently cross-linked with proteins, such asenzymes and antibodies and antigens. Electrochemical tests of thecross-linked redox polymer films showed excellent catalytic activitytowards the oxidation of substrate in solution, high sensitivity, goodreproducibility and stability, thus indicating that it is suitable to beused as sensing membrane in biosensors.

In separate experiments, it was also shown that glucose oxidase andPVFcAA can be readily and homogeneously dispersed into thenanoparticulate alumina together with the hydrophilic PVPAA binder, andthe resulting membrane produced a typical catalytic oxidation currentfor glucose. The experimental results showed that the mediator retainedits fast electron transfer properties and the GOX retained its catalyticactivity after they were screen-printed onto the carbon electrode. theyalso demonstrated that this biosensor has good sensitivity and stabilityfor blood sugar monitoring with a blood sample volume of as little as0.20 μl. The use of screen-printing techniques in the fabrication of thebiosensor enables easy and low-cost mass production. These biosensorcharacteristics are promising for development of miniature glucosebiosensors of high commercial values.

1. A sensor for determining the presence of an analyte in a test sample,said sensor comprising: a nanoparticulate membrane comprisingnanoparticles of at least one inorganic oxide of an element selectedfrom Group IA, IIA, IIIA, IVA, IB, IIB, IIIB, IVAB, VB, VIB, VIIB orVIIIB of the Periodic Table, and wherein an oxidoreductase and anelectrochemical activator are diffusibly dispersed in saidnanoparticulate membrane.
 2. The sensor according to claim 1, whereinthe oxidoreductase is selected from the group consisting of glucoseoxidase, hydrogen peroxidase, horseradish peroxidase, xanthine oxidase,cholesterol oxidase, hydrogen hydrogenase, lactate dehydrogenase,glucose dehydrogenase, NADH dehydrogenase, sarcosine oxidase, lactateoxidase, alcohol dehydrogenase, hydroxybutyrate dehydrogenase, glyceroldehydrogenase, sorbitol dehydrogenase, malate dehydrogenase, galactosedehydrogenase, malate oxidase, galactose oxidase, xanthinedehydrogenase, alcohol oxidase, choline oxidase, xanthine oxidase,choline dehydrohenase, pyruvate dehydrogenase, pyruvate oxidase, oxalateoxidase, bilirubin oxidase, glutamate dehydrogenase, glutamate oxidase,amine oxidase, NADPH oxidase, urate oxidase, cytochrome C oxidase, andactechol oxidase.
 3. The sensor of claim 1, wherein the electrochemicalactivator is a polymeric redox mediator capable of transferringelectrons between the analyte and an electrode present in the sensor. 4.The sensor according to claim 3, wherein the oxidoreductase iscovalently linked to the polymeric redox mediator by cross-linkages. 5.The sensor according to claim 1, wherein the element selected from GroupIA, IIA, IIIA, IVA, IB, IIB, IIIB, IVAB, VB, VIB, VIIB or VIIIB of thePeriodic Table is selected from the group consisting of aluminum,silicon, magnesium and zinc.
 6. The sensor according to claim 1, whereinthe thickness of the membrane ranges from 250 to 500 um.
 7. The sensoraccording to claim 6, wherein the size of the nanoparticles ranges from10 nm to 1 um.
 8. The sensor according to claim 1, wherein the membranefurther comprises a polymeric binder.
 9. The sensor according to claim8, wherein the polymeric binder is a polymer or copolymer comprisingmonomer units selected from the group consisting of vinyl pyridine,vinyl imidazole, acrylamide, acrylonitrile, and acrylhydrazide andacrylic acid.
 10. The sensor according to claim 1, further comprising: achamber for holding the test sample, said chamber being bounded at leastbetween a working area on a working electrode and a working area on areference electrode, wherein the oxidoreductase and the electrochemicalactivator is coated on the working area of the working electrode. 11.The sensor according to claim 10, wherein the working electrodecomprises a material selected from the group consisting of gold, carbon,platinum, ruthenium dioxide, palladium, and conductive epoxies.
 12. Anelectrically non-conductive, nanoparticulate membrane comprisingnanoparticles of at least one inorganic oxide of an element selectedfrom Group IA, IIA, IIIA, IVA, IB, IIB, IIIB, IVAB, VB,VIB, VIIB orVIIIB of the Periodic Table, and wherein an oxidoreductase enzyme and anelectrochemical activator are diffusibly dispersed in saidnanoparticulate membrane.
 13. The membrane of claim 12, wherein theelectrochemical activator is a polymeric redox mediator capable oftransferring electrons.
 14. The membrane of claim 12, wherein theelement selected from Group IA, IIA, IIIA, IVA, IB, IIB, IIIB, IVAB, VB,VIB, VIIB or VIIIB of the Periodic Table is selected from the groupconsisting of aluminum, silicon, magnesium and zinc.
 15. The membrane ofclaim 12, wherein the thickness of the membrane ranges from 250 to 500pm.
 16. The membrane of claim 12, wherein the size of the nanoparticlesranges from 10 nm to 1 um.
 17. The membrane of claim 12, wherein themembrane further comprises a polymeric binder.
 18. The sensor accordingto claim 17, wherein the polymeric binder is a polymer or copolymercomprising monomer units selected from the group consisting of vinylpyridine, vinyl imidazole, acrylamide, acrylonitrile, and acrylhydrazideand acrylic acid.
 19. A process for producing a non-conductive,nanoparticulate membrane, said process comprising: mixing anelectrochemical redox mediator with an oxidoreductase and nanoparticlesof an inorganic oxide of an element from Group IA, IIA, IIIA, IVA, IB,IIB, IIIB, IVAB, VB, VIB, VIIB or VIIIB of the Periodic Table to form ananocomposite ink; and applying said nanocomposite ink onto a substrate.20. The process of claim 19, wherein said nanocomposite ink is appliedaccording to a predetermined pattern.
 21. The process of claim 20,wherein said nanocomposite ink is applied by screen-printing.
 22. Theprocess according to claim 19, wherein the mixing further comprisesmixing a polymeric binder into the nanocomposite ink.
 23. The processaccording to claim 19, wherein the concentration of the electrochemicalactivator in the nanocomposite ink is about 15 mg/ml.
 24. The processaccording to claim 19, wherein the concentration of enzyme in thenanocomposite ink is about 0.2 mg/ml. 25.-44. (canceled)
 45. The sensoraccording to claim 1, wherein the sensor is for determination of glucoseconcentration.